Knockdown of TPI in human dermal microvascular endothelial cells and its impact on angiogenesis in vitro.

INTRODUCTION: Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs. OBJECTIVE: The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined. METHODS: Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored. RESULTS: Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis. CONCLUSION: Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.

Triosephosphate isomerase 1 may be a risk predictor in laryngeal squamous cell carcinoma: a multi-centered study integrating bulk RNA, single-cell RNA, and protein immunohistochemistry.

BACKGROUND: Although great progress has been made in anti-cancer therapy, the prognosis of laryngeal squamous cell carcinoma (LSCC) patients remains unsatisfied. Quantities of studies demonstrate that glycolytic reprograming is essential for the progression of cancers, where triosephosphate isomerase 1 (TPI1) serves as a catalytic enzyme. However, the clinicopathological significance and potential biological functions of TPI1 underlying LSCC remains obscure. METHODS: We collected in-house 82 LSCC tissue specimens and 56 non-tumor tissue specimens. Tissue microarrays (TMA) and immunohistochemical (IHC) experiments were performed. External LSCC microarrays and bulk RNA sequencing data were integrated to evaluate the expression of TPI1. We used a log-rank test and the CIBERSORT algorithm to assess the prognostic value of TPI1 and its association with the LSCC microenvironment. Malignant laryngeal epithelial cells and immune-stromal cells were identified using inferCNV and CellTypist. We conducted a comprehensive analysis to elucidate the molecular functions of TPI1 in LSCC tissue and single cells using Pearson correlation analysis, high dimensional weighted gene co-expression analysis, gene set enrichment analysis, and clustered regularly interspaced short palindromic repeats (CRISPR) screen. We explored intercellular communication patterns between LSCC single cells and immune-stromal cells and predicted several therapeutic agents targeting TPI1. RESULTS: Based on the in-house TMA and IHC analysis, TPI1 protein was found to have a strong positive expression in the nucleus of LSCC cells but only weakly positive activity in the cytoplasm of normal laryngeal cells (p < 0.0001). Further confirmation of elevated TPI1 mRNA expression was obtained from external datasets, comparing 251 LSCC tissue samples to 136 non-LSCC tissue samples (standardized mean difference = 1.06). The upregulated TPI1 mRNA demonstrated a high discriminative ability between LSCC and non-LSCC tissue (area under the curve = 0.91; sensitivity = 0.87; specificity = 0.79), suggesting its potential as a predictive marker for poor prognosis (p = 0.037). Lower infiltration abundance was found for plasma cells, naïve B cells, monocytes, and neutrophils in TPI-high expression LSCC tissue. Glycolysis and cell cycle were significantly enriched pathways for both LSCC tissue and single cells, where heat shock protein family B member 1, TPI1, and enolase 1 occupied a central position. Four outgoing communication patterns and two incoming communication patterns were identified from the intercellular communication networks. TPI1 was predicted as an oncogene in LSCC, with CRISPR scores less than -1 across 71.43% of the LSCC cell lines. TPI1 was positively correlated with the half maximal inhibitory concentration of gemcitabine and cladribine. CONCLUSIONS: TPI1 is dramatically overexpressed in LSCC than in normal tissue, and the high expression of TPI1 may promote LSCC deterioration through its metabolic and non-metabolic functions. This study contributes to advancing our knowledge of LSCC pathogenesis and may have implications for the development of targeted therapies in the future.

Human Triosephosphate Isomerase Is a Potential Target in Cancer Due to Commonly Occurring Post-Translational Modifications.

Cancer involves a series of diseases where cellular growth is not controlled. Cancer is a leading cause of death worldwide, and the burden of cancer incidence and mortality is rapidly growing, mainly in developing countries. Many drugs are currently used, from chemotherapeutic agents to immunotherapy, among others, along with organ transplantation. Treatments can cause severe side effects, including remission and progression of the disease with serious consequences. Increased glycolytic activity is characteristic of cancer cells. Triosephosphate isomerase is essential for net ATP production in the glycolytic pathway. Notably, some post-translational events have been described that occur in human triosephosphate isomerase in which functional and structural alterations are provoked. This is considered a window of opportunity, given the differences that may exist between cancer cells and their counterpart in normal cells concerning the glycolytic enzymes. Here, we provide elements that bring out the potential of triosephosphate isomerase, under post-translational modifications, to be considered an efficacious target for treating cancer.

Molecular identification and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in human immunodeficiency virus/acquired immunodeficiency syndrome patients in Shanghai, China.

BACKGROUND: Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients. METHODS: We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp.; the triosephosphate isomerase, ß-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene. RESULTS: Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and ß-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes (D, Type IV, EbpC, Peru11, EbpD, A, and I) were identified in E. bieneusi-positive specimens. CONCLUSIONS: Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.

Newly discovered roles of triosephosphate isomerase including functions within the nucleus.

Triosephosphate isomerase (TPI) is best known as a glycolytic enzyme that interconverts the 3-carbon sugars dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). TPI is an essential enzyme that is required for the catabolism of DHAP and a net yield of ATP from anaerobic glucose metabolism. Loss of TPI function results in the recessive disease TPI Deficiency (TPI Df). Recently, numerous lines of evidence suggest the TPI protein has other functions beyond glycolysis, a phenomenon known as moonlighting or gene sharing. Here we review the numerous functions ascribed to TPI, including recent findings of a nuclear role of TPI implicated in cancer pathogenesis and chemotherapy resistance.

LKB1-Dependent Regulation of TPI1 Creates a Divergent Metabolic Liability between Human and Mouse Lung Adenocarcinoma.

KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD), and activating mutations frequently co-occur with loss-of-function mutations in TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered comutation is highly aggressive in mouse lung adenocarcinoma (mLUAD). Here, we provide a mechanistic explanation for this difference by uncovering an evolutionary divergence in the regulation of triosephosphate isomerase (TPI1). In hLUAD, TPI1 activity is regulated via phosphorylation at Ser21 by the salt inducible kinases (SIK) in an LKB1-dependent manner, modulating flux between the completion of glycolysis and production of glycerol lipids. In mice, Ser21 of TPI1 is a Cys residue that can be oxidized to alter TPI1 activity without a need for SIKs or LKB1. Our findings suggest this metabolic flexibility is critical in rapidly growing cells with KRAS and TP53 mutations, explaining why the loss of LKB1 creates a liability in these tumors. SIGNIFICANCE: Utilizing phosphoproteomics and metabolomics in genetically engineered human cell lines and genetically engineered mouse models (GEMM), we uncover an evolutionary divergence in metabolic regulation within a clinically relevant genotype of human LUAD with therapeutic implications. Our data provide a cautionary example of the limits of GEMMs as tools to study human diseases such as cancers. This article is highlighted in the In This Issue feature, p. 799.

[Prevalence and risk factors of Giardia lamblia infections among colorectal cancer patients in Henan Province].

OBJECTIVE: To investigate the epidemiological characteristics and identify the risk factors of Giardia lamblia infections among patients with colorectal cancer in Henan Province. METHODS: A cross-sectional study was performed for questionnaire surveys among colorectal cancer patients in Henan Cancer Hospital during the period from March to July, 2021. Patients' stool samples were collected, and the triosephosphate isomerase (tpi) gene of G. lamblia was amplified in stool samples using nested PCR assay to characterize the parasite genotype. Univariate analysis and multivariate logistic regression analyses were employed to identify the risk factors of G. lamblia infections among colorectal cancer patients. RESULTS: A total of 307 colorectal cancer patients were investigated, including 176 males (57.3%) and 131 females (42.7%). PCR assay detected 8.1% [95% confidential interval (CI): (0.056, 0.117)] prevalence of G. lamblia infections among the study subjects, and there was no significant difference in the prevalence between men [9.1%, 95% CI: (0.057, 0.143)] and women [6.9%, 95% CI: (0.037, 0.125)] (χ2 = 0.495, P = 0.482). In addition, there was no age-specific prevalence of G. lamblia infections among the participants (χ2 = 1.534, P = 0.675). Multivariate logistic regression analysis identified use of septic tanks [odds ratio (OR) = 3.336, 95% CI: (1.201, 9.267)], daily use of well water [OR = 3.042, 95% CI: (1.093, 8.465)] and raising livestock [OR = 3.740, 95% CI: (1.154, 12.121)] as risk factors of G. lamblia infections among colorectal cancer patients, and the prevalence of abdominal pain was significantly greater in colorectal cancer patients with G. lamblia infections than in those without infections (P = 0.017). Among the 25 patients with G. lamblia infections, assemblage A was characterized in 24 (96.0%) cases and assemblage B in one case (4.0%). CONCLUSIONS: The prevalence of G. lamblia is high among colorectal cancer patients in Henan Province, and assemblage A is the dominant genotype of G. lamblia. Use of septic tanks, daily use of well water and raising livestock are risk factors of G. lamblia infections among patients with colorectal cancer.

Occurrence, genetic characterization, and zoonotic importance of Giardia duodenalis in various species of rodents (Mus musculus, Rattus norvegicus, and Rattus rattus).

Giardia duodenalis is a well-known flagellated parasite and the causative agent of protozoal diarrhea in animals and humans worldwide. Current study was aimed at determination of G. duodenalis prevalence, genetic variation and zoonotic significance in various species of rodents in Shiraz, southwestern Iran. In brief, 120 fecal specimens were collected from rodents (Rattus rattus, Rattus norvegicus, and Mus musculus) during May up to November 2021 and microscopically examined for Giardia cysts. Further molecular characterization of positive samples was done by nested-PCR, followed by nucleotide sequencing of the triose phosphate isomerase (tpi) gene. A total prevalence of 3.3% (4/120) was observed in rodents, with highest rate in black rats [5% (2/40)]. Regarding brown rats and house mice, only one sample was found to be positive, showing 2.5% and 2.5% prevalence, respectively. It is noteworthy that Giardia B and G assemblages were found in black rats (one case/genotype), whereas the only positive samples from brown rats and house mice were characterized as assemblage G. The major findings of the present study were the presence of both zoonotic and non-zoonotic Giardia assemblages in examined rats in Shiraz and the potential of black rats to harbor Giardia infection to humans. These concerns should be taken seriously in terms of public health. Nevertheless, the true epidemiology and assemblage distribution of Giardia is still open to question.

Elevated nuclear localization of glycolytic enzyme TPI1 promotes lung adenocarcinoma and enhances chemoresistance.

Increased glycolysis is a hallmark of tumor, which can provide tumor cells with energy and building blocks to promote cell proliferation. Recent studies have shown that not only the expression of glycolytic genes but also their subcellular localization undergoes a variety of changes to promote development of different types of tumors. In this study, we performed a comprehensive analysis of glycolysis and gluconeogenesis genes based on data from TCGA to identify those with significant tumor-promoting potential across 14 types of tumors. This analysis not only confirms genes that are known to be involved in tumorigenesis, but also reveals a significant correlation of triosephosphate isomerase 1 (TPI1) with poor prognosis, especially in lung adenocarcinoma (LUAD). TPI1 is a glycolytic enzyme that interconverts dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP). We confirm the upregulation of TPI1 expression in clinical LUAD samples and an inverse correlation with the overall patient survival. Knocking down of TPI1 in lung cancer cells significantly reduced cell migration, colony formation, and xenograft tumor growth. Surprisingly, we found that the oncogenic function of TPI1 depends on its translocation to cell nucleus rather than its catalytic activity. Significant accumulation of TPI1 in cell nucleus was observed in LUAD tumor tissues compared with the cytoplasm localization in adjacent normal tissues. Moreover, nuclear translocation of TPI1 is induced by extracellular stress (such as chemotherapy agents and peroxide), which facilitates the chemoresistance of cancer cells. Our study uncovers a novel function of the glycolytic enzyme TPI1 in the LUAD.

TPI1-reduced extracellular vesicles mediated by Rab20 downregulation promotes aerobic glycolysis to drive hepatocarcinogenesis.

Rab GTPases are major mediators that ensure the proper spatiotemporal regulation of intracellular trafficking. Functional impairment and altered expression of Rab proteins have been revealed in various human cancers. There is an emerging evidence about the role of Rab proteins in the biogenesis of extracellular vesicles (EVs). In hepatocellular carcinoma (HCC), using RNA sequencing comparing expression profiles of adjacent non-tumorous tissues and HCC, Rab20 is identified to be the most frequently downregulated Rab member in HCC. Functionally, restoration of Rab20 in metastatic HCC cells results in the release of EVs with a diminished activity to promote cell growth, motility and metastasis. Conversely, EVs released from normal liver cells with Rab20 knockdown loses suppressive effect on HCC cell growth and motility. Proteomic profiling revealed the level of triosephosphate isomerase 1 (TPI1), a glycolytic enzyme, in EVs to be positively associated with Rab20 expression of the releasing cells. TPI1 targeted to be expressed in EVs released by Rab20 knockdown cells compromises the oncogenic activity of EVs. Besides, EVs released by TPI1 knockdown cells recapitulates the promoting effect of EVs derived from HCC cells with Rab20 underexpression. Aerobic glycolysis is beneficial to the survival and proliferation of tumour cells. Here, we observed that the enhanced cell growth and motility are driven by the enhanced aerobic glycolysis induced by EVs with reduced TPI1. The addition of glycolytic inhibitor blocks the promoting effect of EVs with reduced TPI1. Taken together, our study provides a mechanistic link among tumour cell-derived EVs and glucose metabolism in HCC with Rab20 deregulation.

Glycolysis- and immune-related novel prognostic biomarkers of Ewing's sarcoma: glucuronic acid epimerase and triosephosphate isomerase 1.

INTRODUCTION: Owing to the poor prognosis of Ewing's sarcoma, reliable prognostic biomarkers are highly warranted for clinical diagnosis of the disease. MATERIALS AND METHODS: A combination of the weighted correlation network analysis and differentially expression analysis was used for initial screening; glycolysis-related genes were extracted and subjected to univariate Cox, LASSO regression, and multivariate Cox analyses to construct prognostic models. The immune cell composition of each sample was analysed using CIBERSORT software. Immunohistochemical analysis was performed for assessing the differential expression of modelled genes in Ewing's sarcoma and paraneoplastic tissues. RESULTS: A logistic regression model constructed for the prognosis of Ewing's sarcoma exhibited that the patient survival rate in the high-risk group is much lower than in the low-risk group. CIBERSORT analysis exhibited a strong correlation of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages (P < 0.05). Immunohistochemical analysis confirmed the study findings. CONCLUSIONS: GLCE and TPI1 can be used as prognostic biomarkers to predict the prognosis of Ewing's sarcoma, and a close association of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages provides a novel approach to the disease immunotherapy.

Metabolic Enzyme Triosephosphate Isomerase 1 and Nicotinamide Phosphoribosyltransferase, Two Independent Inflammatory Indicators in Rheumatoid Arthritis: Evidences From Collagen-Induced Arthritis and Clinical Samples.

Metabolic intervention is a novel anti-rheumatic approach. The glycolytic regulator NAMPT has been identified as a therapeutic target of rheumatoid arthritis (RA), while other metabolic regulators coordinating NAMPT to perpetuate inflammation are yet to be investigated. We continuously monitored and validated expression changes of Nampt and inflammatory indicators in peripheral while blood cells from rats with collagen-induced arthritis (CIA). Gene transcriptional profiles of Nampt+ and Nampt++ samples from identical CIA rats were compared by RNA-sequencing. Observed gene expression changes were validated in another batch of CIA rats, and typical metabolic regulators with persistent changes during inflammatory courses were further investigated in human subjects. According to expression differences of identified genes, RA patients were assigned into different subsets. Clinical manifestation and cytokine profiles among them were compared afterwards. Nampt overexpression typically occurred in CIA rats during early stages, when iNos and Il-1ß started to be up-regulated. Among differentially expressed genes between Nampt+ and Nampt++ CIA rat samples, changes of Tpi1, the only glycolytic enzyme identified were sustained in the aftermath of acute inflammation. Similar to NAMPT, TPI1 expression in RA patients was higher than general population, which was synchronized with increase in RFn as well as inflammatory monocytes-related cytokines like Eotaxin. Meanwhile, RANTES levels were relatively low when NAMPT and TPI1 were overexpressed. Reciprocal interactions between TPI1 and HIF-1α were observed. HIF-1α promoted TPI1 expression, while TPI1 co-localized with HIF-1α in nucleus of inflammatory monocytes. In short, although NAMPT and TPI1 dominate different stages of CIA, they similarly provoke monocyte-mediated inflammation.

Giardia duodenalis multi-locus genotypes in dogs with different levels of synanthropism and clinical signs.

BACKGROUND: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. METHODS: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. RESULTS: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. CONCLUSION: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage.

Proteomics analysis identified TPI1 as a novel biomarker for predicting recurrence of intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is the second most common tumor in primary liver cancer, but the prognostic factors associated with long-term outcomes after surgical resection remain poorly defined. This study aimed to develop a novel prognostic classifier for patients with ICC after surgery. METHODS: Using a proteomics approach, we screened tumor markers that up-regulated in ICC tissues, and narrowed down by bioinformatics analysis, western blot and immunohistochemistry. Prognostic markers were identified using Cox regression analyses in primary training cohort and the predictive models for time to recurrence (TTR) were established. The predictive accuracy of predictive model was validated in external validation cohort and prospective validation cohort. MTT assay, clonal formation assay and trans-well assays were used to verify the effect on the proliferation and migration in ICC cell line. RESULTS: Triosephosphate isomerise (TPI1) was significantly up-regulated in ICC tissues and Kaplan-Meier analysis reveals that higher TPI1 expression was strongly correlated with higher recurrence rate of ICC patients. In the primary training cohort, mean TTR was significantly longer (p < 0.0001) than in the low-risk group (26.9 months for TTR, 95% CI 22.4-31.5) than in the high-risk group (14.5 months for TTR, 95% CI 10.6-18.4). Similar results were observed in two validation cohorts. In addition, a nomogram to predict recurrence was developed. Moreover, Knockdown of TPI1 by shRNA inhibited ICC cell growth, colony information, migration, invasion in vitro. CONCLUSIONS: Current prognostic models were accurate in predicting recurrence for ICC patients after surgical resection.

First multilocus sequence typing (MLST) of Giardia duodenalis isolates from humans in Romania.

BACKGROUND: Giardia duodenalis is one of the most prevalent and highly diverse human parasites, encompassing a complex of eight genetically distinct assemblages, each further divided into sub-assemblages. While in recent years, G. duodenalis genotype distribution patterns in humans have been intensely studied, there is still very little information available on the diversity of Giardia genotypes and sub-assemblages infecting people in Romania. In the present study, we investigated the genetic diversity of Giardia duodenalis in asymptomatic patients from Romania. METHODS: Over an 11-month period, human feces from 7805 healthy adults were screened by microscopic analysis for G. duodenalis cysts during their obligatory periodic check-ups. DNA extraction was performed from microscopic-positive fecal samples, followed by multilocus sequence typing of four genetic loci of the ITS region, gdh, tpi and bg genes, followed by DNA sequencing and phylogenetic analysis. Statistical analysis was performed using EpiInfo 2000 software. RESULTS: The prevalence of giardiasis in the present study was 0.42% (33/7805). Twenty-three samples (76.67%) were successfully genotyped at each locus. The bg and tpi genes had the highest typing success rate (100%). The identified assemblages were assemblage A in 27 cases (subtypes A2 and A3), and B in 3 cases. CONCLUSIONS: To our knowledge, the present study is the first report of multilocus sequence typing of G. duodenalis isolated from humans in Romania. The present results may shed light on G. duodenalis infection in humans at a regional and national level, thus increasing awareness against this parasitic infection.

Occurrence and molecular characterization of Giardia duodenalis in lambs in Djelfa, the central steppe of Algeria.

Little is known of the prevalence and genetic identity of Giardia duodenalis in sheep in Algeria. The present study aimed at characterizing G. duodenalis in lambs up to 6 months of age in Djelfa, Algeria. A total of 346 fecal specimens were collected from 28 farms and screened for G. duodenalis cysts by zinc sulfate flotation microscopy, and positive specimens were confirmed using a direct immunofluorescence assay. Microscopy-positive specimens were analyzed by PCR and sequence analysis of the triosephosphate isomerase and glutamate dehydrogenase genes to determine G. duodenalis assemblages. Coprological examination indicated that the overall infection rate was 7.0% (24/346). Lambs under 3 months of age had higher infection rate (18/197, 9.0%) than older (6/149, 4.0%) animals, and animals with diarrhea (7/44, 16.0%) had higher infection rate than animals without diarrhea (17/302, 5.6%). PCR sequence analyses of the 15 G. duodenalis isolates revealed the presence of assemblages A in 6 isolates, assemblage E in 7 isolates, and both in 2 isolates. Assemblage A was only found in pre-weaned lambs with diarrhea, while assemblage E was mostly found in post-weaned lambs without diarrhea. The assemblage E isolates from sheep were genetically related to those from cattle in Algeria, while assemblage A isolates were from a well-known subtype prevalent in humans. Data generated from the study improve our understanding of the transmission of G. duodenalis in Algeria.

Multilocus genotyping of Giardia duodenalis in Southwestern Iran. A community survey.

Giardia duodenalis is one of the main enteric pathogens associated with diarrheal disease. In developing countries, giardiasis is a major public health concern, particularly in children under five years of age. This study aimed to evaluate the occurrence and genetic diversity of G. duodenalis causing human infections in Shushtar County, Southwestern Iran. Individual faecal specimens were collected from 1,163 individuals (male/female ratio: 0.9; age range 2-75 years) with (n = 258) and without (n = 905) gastrointestinal symptoms living in rural and urban settings during the period 2017-2018. Conventional (sucrose flotation and microscopy) methods were used for the initial detection of G. duodenalis cysts in faecal specimens. Microscopy-positive samples were confirmed by PCR amplification and sequencing of the small subunit rRNA (ssu rRNA) gene of the parasite. A multilocus genotyping (MLG) scheme targeting the triose phosphate isomerase (tpi), the glutamate dehydrogenase (gdh), and the beta-giardin (bg) genes was used for genotyping purposes. Giardia duodenalis cysts were detected in 7.7% (90/1,163) of samples by microscopy, of which 82 were confirmed by ssu-PCR. Successful amplification and sequencing results were obtained for 23.2% (19/82), 9.8% (8/82), and 8.5% (7/82) of the confirmed samples at the tpi, gdh, and bg loci, respectively. MLG data for the three loci were available for two samples only. Out of the 24 samples genotyped at any loci, 50% (12/24) were identified as assemblage A and the remaining half as assemblage B. Overall, AII was the most prevalent sub-assemblage detected (41.7%, 10/24), followed by BIII (25.0%, 6/24), discordant BIII/BIV (5/24) or AII/AIII (2/24) sequences, and BIV (1/24). No significant correlation was demonstrated between a given assemblage/sub-assemblage and the occurrence of clinical symptoms. No genotypes adapted to animal hosts other than humans (e.g. assemblages C-F) were found circulating in the investigated human population, suggesting that transmission of human giardiasis in this Iranian region is primarily of anthroponotic nature. Further molecular-based studies are needed to confirm and expand these results, and to ascertain the presence and public health relevance of the parasite in environmental (e.g. drinking water) samples.

Concordance of Giardia duodenalis assemblages determined by different PCR methodologies in three observational studies in Cuba.

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates.

Molecular characterization of zoonotic Cryptosporidium spp. and Giardia duodenalis pathogens in Algerian sheep.

Little is known about the presence of Cryptosporidium spp. and Giardia duodenalis in Algerian sheep, nor their potential role as zoonotic reservoirs. This study aimed to investigate the occurrence and distribution of these two protists in lambs. A total of 83 fecal samples were collected from lambs (< 40 days old) from 14 different farms. Samples were screened for Cryptosporidium spp. and Giardia duodenalis presence using immunofluorescent techniques (IF). Nested PCR of the small subunit ribosomal RNA (rRNA) gene, followed by restriction fragment length polymorphism (PCR-RFLP) and sequence analyses were used to identify Cryptosporidium species. C. parvum was further subtyped by sequencing the highly polymorphic 60 kDa glycoprotein (gp60) gene. For G. duodenalis, nested PCR of the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes was performed and then PCR-RFLP was used to identify G. duodenalis assemblages. Cryptosporidium oocysts and Giardia cysts were detected in 36/83 (43%) and 23/83 (28%) of fecal samples, respectively. Of the 21/36 (58%) Cryptosporidium samples that were positive with IF, 16/21 (76%) were identified as C. parvum, and 5/21 (24%) as C. ubiquitum. From 15C. parvum isolates, 2 subtypes were identified within the IIa subtype family, including IIaA21G2R1 (3/15) and IIaA13G2R1 (1/15), while IIdA16G1 (11/15) was the only subtype identified from the IId subtype family. Of the 16/23 (69%) G. duodenalis IF-positive samples, the most frequent assemblage was ruminant-specific assemblage E (10/16), followed by assemblage D (4/16), and A + E mixed assemblages (2/16). This study is the first to identify and genotype both Cryptosporidium spp. and Giardia duodenalis in Algerian lambs, and is also the first to describe G. duodenalis assemblage D in small ruminants. The presence of zoonotic C. parvum subtype families (IIa, IId), C. ubiquitum, as well as G. duodenalis assemblage A + E, indicates that sheep could play an important role as a potential reservoir for protists.

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox-based modifications.

Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI-Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI-Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI-Cys13, mutations in AtcTPI-Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI-Cys13 and AtcTPI-Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI-Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent-exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S-glutathionylation protects AtcTPI from irreversible chemical modifications and re-routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.